桃叶片总蛋白双向电泳技术的研究

采用改进的O'Farrell双向电泳系统,研究适于桃树叶片蛋白质组分析的双向电泳方法。通过对不同蛋白样品提取方法、不同pH梯度范围、不同上样量的比较,探索出一套适合桃树叶片总蛋白分析的双向电泳方法,即以TCA/丙酮沉淀法提取叶片蛋白质样品,采用两性电解质配比pH3～10︰pH5～7为1∶4的IEF胶条,按90μg蛋白上样量,并结合适宜的双向电泳参数,可获得背景清晰、重复性较好、蛋白分辨率高的双向电泳图谱。经此方法分离的桃叶片蛋白质经串联质谱分析(MS/MS)得到了较好的鉴定。

Study of two-dimensional gel electrophoresis for total proteins from peach leaves

Two-dimensional gel electrophoresis(2-DE) was used to seek out the procedure suitable for leaf proteome analysis of peach.Two sample preparation methods,two different pH gradients and three different sample amounts were compared.The result showed that good 2-DE gels obtained by the procedure of TCA/acetone precipitation,ampholine pH3～10∶pH 5～7 in IEF gels according the ratio of 1∶4,90 μg loaded sample combined with suitable electrophoresis parameter.This improved 2-DE system resulted in a protein profile of better resolution and higher protein yields as analyzed by Image Master 2D software.Good results were also obtained by MS/MS.